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LFD-RPA primer and probe combination for visually detecting phytophthora colocasia and detection method thereof

A technology of LF-RPA and detection primers, applied in the biological field, can solve problems such as no relevant application reports, and achieve the effects of good amplification effect, good practicability and strong band specificity

Pending Publication Date: 2021-01-29
INST OF PLANT PROTECTION FAAS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, LFD-RPA detection is mainly used in the detection of human and animal pathogens, food safety and environmental sanitation, but there are few reports on the detection of plant pathogenic oomycetes, especially for the detection of Phytophthora taro at home and abroad.

Method used

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  • LFD-RPA primer and probe combination for visually detecting phytophthora colocasia and detection method thereof
  • LFD-RPA primer and probe combination for visually detecting phytophthora colocasia and detection method thereof
  • LFD-RPA primer and probe combination for visually detecting phytophthora colocasia and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Embodiment 1 Phytophthora taro LFD-RPA detects the design of primers and probes

[0045] (1) Acquisition of Ypt gene from Phytophthora colocasiae Racib.

[0046] ① Extraction of Genomic DNA from Phytophthora taro

[0047] Adopt CTAB method to extract Phytophthora taro genome DNA, concrete process is as follows: get the mycelia powder after 50mg freeze-drying in 1.5ml centrifuge tube, add 900 μ l 2% CTAB (hexadecyltrimethylammonium bromide) extraction solution ( 2% CTAB; 100mmol / LTris-HCl (tris-hydroxymethylaminomethane hydrochloride), pH 8.0; 20mmol / L EDTA (disodium ethylenediaminetetraacetic acid), pH 8.0; 1.4mol / L NaCl) and 90μl 10% SDS (sodium dodecylbenzenesulfonate) and mix well, put in water bath at 55~60℃ for 1.5h, shake and mix once every 10min, centrifuge (12,000rpm) for 15min after 1.5h in water bath, take the supernatant and add Phenol / chloroform / isoamyl alcohol (the volume ratio of phenol, chloroform and isoamyl alcohol is 25:24:1) equal to the volume of t...

Embodiment 2

[0062] Example 2Visual detection of LFD-RPA primer and probe combination to Phytophthora taro

[0063] (1) Extraction of Phytophthora taro Genomic DNA:

[0064] Genomic DNA of Phytophthora taro was extracted by the CTAB method in Example 1, and the DNA concentration was detected by an ultraviolet spectrophotometer and diluted to 100 ng / μl for use.

[0065] (2) LFD-RPA reaction system: the reaction system is 50 μl, including 29.5 μl reaction buffer (Rehydration buffer), 10 μM PcoRPALFD-F and 2.1 μl biotin-labeled PcoRPALFD-R, 10 μM PcoRPALFD-P 0.6 μl probe, to be Measure template DNA 2.0 μl, sterile double distilled water 11.2 μl, mix the above components and add to RPA freeze-dried enzyme powder, then add 2.5 μl 280mM magnesium acetate (MgAc) and mix upside down; replace the template DNA with ddH 2 O was used as a negative control.

[0066] (3) LFD-RPA reaction: Incubate the reaction system in step (2) at 39°C for 5 minutes, mix the reaction tube again, and continue to incu...

Embodiment 3

[0069] Embodiment 3 LFD-RPA primer and probe combination are to the specific amplification of Phytophthora taro

[0070] Using 22 strains of Phytophthora taro and 15 other pathogenic fungi and oomycetes from Fujian, Jiangxi and Guangxi in my country as test materials, the specificity of the detection primers and probes was verified by LFD-RPA.

[0071] (1) Tested strains

[0072] serial number species name the host source Number of strains 1 Phytophthora colocasiae Racib. Colocasia esculenta Fujian 15 2 Phytophthora colocasiae Racib. Colocasia esculenta Jiangxi 3 3 Phytophthora colocasiae Racib. Colocasia esculenta Guangxi 4 4 Phytophthora capsici P.capsici Pepper Capsicum frutescent Fujian 1 5 P.cactorum Apple Malus pumila Shaanxi 1 6 Phytophthora infestans P.infestans Potato Solanum tuberosum Fujian 1 7 Phytophthora sojae P.sojae Soy Glycine max Fujian 1 8 Peronophythora litch...

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PUM

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Abstract

The invention discloses an LFD-RPA primer and a probe combination for visually detecting phytophthora colocasia and a detection method of the LFD-RPA primer and the probe combination, and belongs to the technical field of biology. The invention discloses the LFD-RPA detection primer and probe combination for phytophthora colocasia. The LFD-RPA detection primer and the probe combination comprises two primers PcoRPALFD-F, a biotin-labeled PcoRPALFD-R and a probe PcoRPALFD-P. The combination of the LF-RPA detection primer and the probe is amplified at the constant temperature of 39 DEG C and thenis detected by a lateral flow chromatography test strip, and the test strip can be observed to show a positive strip in a detection area. The LF-RPA detection primer and the probe combination and thedetection method thereof, disclosed by the invention, can be used for quickly, sensitively and accurately detecting phytophthora colocastans infected plants in production practice, and can also be used for early diagnosis of field diseases and monitoring and identification of germs, so that reliable technical and theoretical bases are provided for prevention and treatment of diseases caused by phytophthora colocastans.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a combination of LFD-RPA primers and probes for the visual detection of Phytophthora colocea and a detection method thereof. Background technique [0002] Taro (Colocasia esculenta), also known as taro, taro, or taro, is a perennial tuber plant of the genus Colocasia in the family Araceae, but it is often cultivated as an annual plant. It is an important economic crop, mainly in my country. Planted in South China, Southwest China and the Yangtze River Valley. Taro blight is a common disease in taro production, and its distribution range is wide, especially in southern my country. The disease is caused by the infection of Phytophthora colocasiae Racib. It is prevalent from June to September, mainly harming leaves and petioles, and also harming bulbs in severe cases. The disease spots develop rapidly in high temperature and rainy days, causing a large number of leaves to...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6844C12Q1/04C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/6844C12Q2521/507C12Q2521/101C12Q2565/625
Inventor 王荣波李本金刘裴清陈庆河赵玉梅翁启勇
Owner INST OF PLANT PROTECTION FAAS
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